What Is in Fetal Bovine Serum Used for Cell Culture? Integrated Proteomics and Peptidomics Reveal Grade-Dependent Molecular Profiles

Fetal bovine serum (FBS) is a widely used but poorly defined mammalian cell culture supplement, introducing substantial batch-to-batch and grade-specific variability that compromises experimental reproducibility. We performed integrative proteomics and peptidomics profiling of three commercially available FBS grades using data-independent acquisition (DIA) mass spectrometry. A conserved core of 979 proteins shared across all grades was enriched for extracellular matrix organization, immune modulation, and growth factor regulation, explaining FBS's broad utility in standard cell culture. Differential abundance analysis revealed 88 proteins differing significantly between high-grade and low-grade products, while peptidomic profiling detected over 200 differentially abundant native peptide sequences. High-grade FBS was enriched in adhesion proteins (fibronectin, vitronectin, thrombospondin-1) and growth factor binding proteins (IGFBP2, IGFBP3), supporting robust cell attachment and proliferation, while low-grade serum exhibited elevated detoxification and oxidative stress response proteins consistent with a stress-adapted biochemical signature. Multiomics integration via DIABLO revealed coordinated proteolytic regulation distinguishing serum grades, with native peptides demonstrating greater intergrade divergence than intact proteins. These findings establish a molecular framework for rational serum selection and mass spectrometry-guided lot qualification in biotechnology and regenerative medicine, and identify functional modules structural scaffolding, carrier buffering, and bioactive peptide signaling that underpin FBS bioactivity and support the development of chemically defined culture media.